Pentameric ligand-gated ion channels (pLGICs) are neurotransmitter-activated receptors that mediate fast synaptic transmission. In pLGICs, the flexible loops in each subunit that connect the extracellular binding domain (loops 2, 7, 9) to the transmembrane channel domain (M2-M3 loop) are essential for coupling ligand binding to channel gating. Comparing the recent crystal structures of two bacterial pLGIC homologues, ELIC and GLIC, suggests channel opening is associated with rearrangements in these loops but the specifics of these motions remain unknown. Here, using site-directed spin labeling electron paramagnetic resonance (SDSL EPR) spectroscopy, we measured proton-induced movements of loop 2, loop 9, and M2-M3 loop in functional GLIC channels reconstituted into liposomes. The loops undergo significant proton-dependent motion, with loop 2 and loop 9 moving inward toward the channel lumen, and the M2-M3 loop moving at least 5 angstrom outward away from the channel lumen. The movements are consistent with the gating motions predicted by comparing the ELIC and GLIC structures, and suggest the ELIC structure represents the resting state of pLGICs.